DNA repair and free radicals, new insights into the mechanism of spore photoproduct lyase revealed by single amino acid substitution.
The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(alpha-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed.
Chandor-Proust
Alexia
A
Commissariat à l'Energie Atomique (CEA), Institut de Recherches en Technologie et Sciences pour le Vivant, Laboratoire de Chimie et Biologie des Métaux, Grenoble 38054, France.
Berteau
Olivier
O
Douki
Thierry
T
Gasparutto
Didier
D
Ollagnier-de-Choudens
Sandrine
S
Fontecave
Marc
M
Atta
Mohamed
M
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D001412:Bacillus subtilis / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004260:DNA Repair; D005609:Free Radicals; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008956:Models, Chemical; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013441:Sulfinic Acids / Q000737:chemistry; D013941:Thymine / Q000737:chemistry; D014466:Ultraviolet Rays
D000596:Amino Acids; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D011506:Proteins; D013441:Sulfinic Acids; C088014:spore photoproduct lyase; D003545:Cysteine; D000409:Alanine; D013941:Thymine